There are several methods reported for producing L-phenylalanine. In one such method, L-phenylalanine is produced by contacting trans-cinnamic acid and ammonium ions in the presence of the enzyme L-phenylalanine ammonia-lyase (PAL) produced by strains of microorganisms. This procedure is known and described in U.S. Pat. No. 4,584,269, which is directed toward a method for producing L-phenylalanine and preserving the useful life of the enzyme by conducting the reaction under substantially anaerobic conditions. U.S. Pat. No. 4,584,273 describes a process for the production of L-phenylalanine ammonia-lyase by fermentation. The stability and useful life of the enzyme is improved by maintaining the enzyme in anaerobic, static conditions. U.S. Pat. No. 4,574,117 discloses a method for producing L-phenylalanine using PAL. Reducing agents are added to the process to minimize the effects of oxygen on the enzyme.
Yamada, et al., Production of L-Phenylalanine From Trans-Cinnamic Acid With Rhodotorula glutinis Containing L-Phenylalanine Ammonia-lyase Activity, Applied Environmental Microbiology, Vol. 42. p. 773, Aug. 1981, describe the use of Rhodotorula glutinis for the enzymatic conversion of trans-cinnamic acid to L-phenylalanine.
U.K. Pat. No. 1,489,468 (1977) teaches the use of the enzyme to convert trans-cinnamic acid and ammonium ions to L-phenylalanine by adjusting the concentration of the reactants to drive the reaction towards L-phenylalanine production instead of its breakdown.
L-phenylalanine can also be produced by transaminating phenylpyruvate. Calton, et al., in "The Production of L-Phenylalanine By Polyazetidine Immobilized Microbes", Bio/Technology, Vol. 4 (April 1986) describe the production of L-phenylalanine by the transamination of phenylpyruvate using aspartic acid. The ragents are contacted in the presence of a microorganism capable of producing sufficient amounts of the transaminase used to enzymatically catalyze the transamination. Pyruvic acid, or its salts, is a by-product of this method and constitutes a contaminant that must be separated from L-phenylalanine.
Recovery of L-phenylalanine was discussed in Yamada, et al. Yamada describes the use of ion exchange techniques for the separation of L-phenylalanine from a reaction mixture containing L-phenylalanine and L-phenylalanine precursors.
U.K. Pat. No. 1,489,468 (1977) describes the separation of L-phenylalanine from a reaction mixture by use of ion exchange techniques followed by recrystallization. The reference also discloses that L-phenylalanine can be alternately isolated by filtration and azeotropic distillation with benzene.
U.S. Pat. No. 4,584,399 discloses a method for separating L-phenylalanine from an aqueous solution by contacting the solution with activated carbon and then eluting L-phenylalanine from the activated carbon followed by ion exchange to remove further contaminants and then crystallization of L-phenylalanine.
U.S. Pat. No. 4,584,400 describes a process for separating L-phenylalanine and tyrosine by using a nonpolar, porous synthetic adsorbant having a higher affinity for L-phenylalanine than tyrosine, followed by elution of L-phenylalanine from the adsorbant.
E.P. Pat. No. 0140713 (1985) discloses a procedure for the separation of L-phenylalanine by evaporation of a reactant mixture containing L-phenylalanine and L-phenylalanine precursors at reduced pressure and temperature until L-phenylalanine precipitates.
There are disadvantages to all of these separation processes. Separation of L-phenylalanine from a reaction mixture through the use of ion exchange columns or adsorbtion procedures has the disadvantage of being expensive due to the cost of the resins and/or adsorbants. In addition, after elution, one is left with a large volume of dilute solution which is difficult to handle and requires evaporation before further separation and purification can take place.
The process described in E.P. Pat. No. 0140713 is undesirable because it yields L-phenylalanine contaminated with unacceptable amounts of precursors such as trans-cinnamic acid.
Accordingly, there is the need for a purification process which can readily provide L-phenylalanine in substantially pure from and particularly minimize contamination with trans-cinnamic acid or pyruvic acid.